RNA interference (RNAi) technology has recently emerged as a powerful tool to investigate host proteins involved in virus replication on a genome-wide level (Krishnan, M. N.; Ng, A.; Sukumaran, B.; Gilfoy, F. D.; Uchil, P. D.; Sultana, H.; Brass, A. L.; Adametz, R.; Tsui, M.; Qian, F.; Montgomery, R. R.; Lev, S.; Mason, P. W.; Koski, R. A.; Elledge, S. J.; Xavier, R. J.; Agaisse, H.; Fikrig, E., “RNA interference screen for human genes associated with West Nile virus infection,” Nature 2008, v. 455(7210): pp. 242-U67; Brass, A. L.; Dykxhoorn, D. M.; Benita, Y.; Yan, N.; Engelman, A.; Xavier, R. J.; Lieberman, J.; Elledge, S. J., “Identification of host proteins required for HIV infection through a functional genomic screen,” Science 2008, v. 319(5865): pp. 921-926). By systematically silencing >20,000 individual host genes and analyzing their involvement in viral infection, a comprehensive portrait of virus-host interactions can be revealed. The use of this technology has yet to be performed on viral agents requiring BSL-4 biocontainment since traditional high-throughput robotic screening equipment cannot be placed within BSL-4 due to space constraints, aerosol-generation biohazards, and highly restricted access for equipment maintenance. The microelectroporation device described herein addresses these issues through a high-throughput multiplexed microfluidic platform capable of suppressing gene expression using genome-wide RNAi in primary cells upon viral infection.
Two high-throughput formats are currently being used for genome-wide RNAi screening: multiwell plates and microarrays (Carpenter, A. E.; Sabatini, D. M., Systematic genome-wide screens of gene function. Nature Reviews—Genetics, 2004, v. 5(1): pp. 11-22; Erfle, H.; Neumann, B.; Liebel, U.; Rogers, P.; Held, M.; Walter, T.; Ellenberg, J.; Pepperkok, R., “Reverse transfection on cell arrays for high content screening microscopy,” Nature Protocols, 2007, v. 2(2): pp. 392-399). The multiwell-plate approach, where each well contains host cells and a different small-interfering RNA (siRNA), requires microliters of costly RNAi reagents, and comes with bulky, robotic screening equipment that requires regular maintenance. Although a smaller well size is possible, significant well-to-well variation caused by evaporation and temperature gradients limit the screening throughput to 384-well plates. The microarray format consists of printing hundreds of siRNA spots onto a glass slide with a microarray spotter device. Cell transfection is achieved by seeding cells on top of the slide. However, the lack of physical barriers between different spots on these microarrays makes this approach prone to cross-contamination and prevents or greatly impedes analysis of secreted factors. Moreover, seeding the cells in this manner results in wide variability in the number of cells distributed at each RNAi spot.